TW202001244A - Method of aptamer selection and method for purifying biomolecules with aptamers - Google Patents

Abstract

The present invention proposes a method of using nucleic acid aptamers bound with certain disease-associated specific biomolecules to purify the specific biomolecules from patient's specimen samples and to identify biomarkers for the particular disease. The present invention describes a method of using the magnetic assisted rapid aptamer selection (MARAS) to select aptamers having high binding affinity for certain disease-associated biomolecules and not for other non-disease related biomolecules from nucleic acid libraries. The present invention also proposes a method of using the nucleic acid aptamer(s) obtained above as a capture ligand to purify certain disease-associated specific biomolecules from patients' specimen samples. Further, the present invention describes the use of the obtained aptamer as the capture ligands to purify biomolecules within a specific binding affinity range by applying an oscillating magnetic field range as a virtual filter.

Description

Aptamer selection method and method for purifying biomolecules with aptamers

The present invention is generally about finding out positive samples (samples of disease patients) and negative samples (samples of non-disease patients) using the design of positive and negative screening and the structural diversity in the nucleic acid library Difference, and use the magnetic assisted rapid aptamer selection (MARAS) method to screen out the aptamer (aptamer) with the ability to distinguish the difference between the two, and then use the selected aptamer as a capture ligand to obtain positive A method of purifying certain biomolecules associated with aptamer-associated diseases and identifying them in order to discover disease-related biomarkers in attribute samples. Specifically, the present invention relates to a method for screening a nucleic acid library to distinguish the differences between biomolecules contained in disease patients and non-patient samples, and can be used to purify the differences from disease patient samples The screening method of aptamers for capture ligands of disease-related biomolecules, and describes the use of aptamers as capture ligands to purify this differential disease-related biomolecules from disease patient specimens Methods.

Purification of the target protein from the total protein and identification of the target protein must rely on appropriate protein purification techniques to separate the target protein from the total protein for subsequent analysis. Common protein purification techniques are: (1) Membrane separation uses most of the molecular size of the protein to achieve the purpose of separation; (2) Chromatography uses the molecular size, charge, and polarity of the protein The difference with affinity is further divided into: colloidal filtration (Gel Filtration), ion exchange chromatography (Ion-Exchange Chromatography), hydrophobic interaction chromatography (Hydrophobic Interaction Chromatography, HIC), affinity chromatography (Affinity Chromatography ), Partition Chromatography, High-Performance (Pressure) Liquid Chromatography; and (3) Magnetic separation (Magnetic separation) using its own magnetic properties, will Molecules (DNA, antibodies, peptides) that can specifically bind to the target protein, fixed on the surface of the magnetic particle and bound to the target protein, through magnetic separation, using a magnet can quickly achieve the effect of separating the target protein from the total protein . The protein purification methods of (1) and (2) above will be separated when the molecular weight of the target protein is close to the molecular weight of the non-target protein, causing the target protein and non-target protein to be mixed together, resulting in reduced purity, such as a thin film Separation is mostly used as a method for preliminary purification and separation of proteins; (3) Although magnetic separation method can quickly separate the target protein from the total protein, in order to desorb the target protein from the magnetic beads, high concentration salt elution is usually used (elution), the elution step takes time and may cause protein activity to decrease, and magnetic separation methods often require antibodies as a tool for protein binding. Antibodies are costly, difficult to store, and have different titers, resulting in high protein purification costs. It is not easy to promote it for practical use.

In the past, it was used to discover biomarkers related to cancer, such as ELISA, Quantitative Immuno-PCR (QI-PCR), protein microarray, two-dimensional gel electrophoresis combined with mass spectrometry, shut gun proteomics, isotope-code affinity tagging combined with mass spectrometer (ICAT) or image analysis media assisted laser desorption/ionization-image mass spectrometry (MALDI-IMS) and nucleic acid aptamers (nucleic acid aptamer) etc. The above experimental methods all have high-throughput characteristics, and can analyze a large amount of differences in protein or RNA expression levels between tumor patients and non-tumor normal people. But the above experimental methods all require professional technology, consume a lot of manpower, time, consumables and expensive instrument analysis operations. However, when there is no suitable monoclonal antibody with high affinity and specificity or the identity of the target biomolecule is unknown, the above method cannot achieve the purpose of protein purification and thus cannot identify the biomarker.

The adaptation system uses the diversity of the constituent nucleic acids in the nucleic acid library to generate the diversity of the structure formed after the nucleic acid is folded to combine with the target biomolecule, and uses the competition mechanism to screen out the ones with high binding affinity and high specificity for the target biomolecule Aptamers, so the former can be used to replace monoclonal antibodies. However, if the identity of the target biomolecule is unknown, the diversity of the structure formed by the nucleic acid in the nucleic acid library can still be used to screen the specimens with the ability to distinguish different attributes (disease patients and non-disease patients) Aptamers.

The aptamers containing binding pockets bind to a variety of target analytes with high specificity and binding affinity, from small molecules (such as organic molecules, ions, peptides, proteins, nucleic acids), large molecules to whole cells, Viruses, parasites or tissues. Once the sequence of the aptamer for the target analyte is identified, the entire aptamer can be generated by chemical synthesis. In addition, aptamers modified with functional groups increase their stability in various biological applications that may be harmful to nucleic acids. Aptamers are not only potentially good tools for targeting pathogenic and malignant cells or tissues and replacing antibodies, but also for purification, diagnostics, biosensors and anti-infectives. Because aptamers have potential in many ways, an effective selection method has been developed—Magnetic-Assisted Rapid Aptamer Selection (MARAS), which is simple and clear enough to quickly screen for targets The analyte has a suitable aptamer with high binding affinity and specificity. The details of "Magnetic-assisted rapid aptamer selection method" are detailed in the Chinese Invention Patent (ZL 2014 1 0570602.0) issued on March 08, 2017 or published in the Patent Gazette of the Republic of China on July 11, 2017 Invention patent (I591218), which is incorporated herein by reference and forms part of this specification.

MARAS does not require a screening cycle. It is a direct screening method. Its construction is simple, fast, efficient, and can ensure the high binding affinity and specificity of the screening aptamers and target development platform, which can improve the application of aptamers. Level. In the method, magnetic particles combined with target molecules are incubated with a nucleic acid library, and part of the nucleic acids will be combined with the target molecules to form a magnetic particle binding complex. The magnetic particle binding compound in the liquid sample moves under the action of the oscillating magnetic field of MARAS, and the magnetic traction force is exerted on the magnetic particles through the magnetic field, so that the magnetic particle binding compound generates an oscillating motion. When the magnetic particle binding compound moves in the liquid Will produce a viscous force opposite to the direction of motion. Under the interaction of these two forces, the binding of the target molecule bound to the magnetic particle and the aptamer on the target molecule will produce a pulling force, providing nucleic acid The competitive mechanism of ligand screening selects nucleic acids (aptamers) with high binding affinity and specificity for target molecules. At the same time, the binding affinity (or equilibrium dissociation constant) between the aptamer and the target molecule can be selected through the conditions of the applied oscillating magnetic field to obtain an aptamer with the desired binding affinity for the target molecule.

Some aspects of the present invention relate to the use of MARAS to select aptamers with the ability to distinguish disease patient specimens from non-patient specimens. This method utilizes the structural diversity formed after the nucleic acids constituting the nucleic acid library are folded to screen An aptamer that can bind to certain disease-related biomolecules in a positive (disease patient) specimen but does not bind to a non-disease-related biomolecule in a negative (non-disease patient) specimen, and then use the obtained aptamer Aptamers to distinguish between positive and negative samples.

The present invention provides an aptamer selection method that uses biologically functionalized magnetic particles to screen oligonucleotides (aptamers) capable of distinguishing between positive and negative specimens from a nucleic acid library.

Some aspects of the present invention relate to methods for using the obtained aptamers with the ability to distinguish between positive and negative specimens as capture ligands to purify certain disease-related biomolecules from samples with positive attributes.

The invention provides a method for purifying certain disease-related biomolecules from positive attribute samples, which uses biologically functionalized magnetic particles and aptamers as capture ligands to generate purification reagents for certain disease-related biomolecules To purify these disease-related biomolecules from samples with positive attributes.

In order to make the above-mentioned features and advantages of the present invention more obvious and understandable, the embodiments are specifically described below in conjunction with the accompanying drawings for detailed description as follows.

The content of the present invention mainly includes two major parts, which are the selection of aptamers with the ability to distinguish positive specimens (disease specimens) and negative specimens (non-disease specimens) from the nucleic acid library by the MARAS method and to The screened aptamers are used as capture ligands to purify disease-related biomarkers from samples with positive attributes. The two parts are described in detail below.

Some aspects of the present invention relate to the use of the structural diversity generated by folding of the diversity of the constituent nucleic acids in the nucleic acid library to screen out the positive samples (diseased patient samples) and negative samples from the nucleic acid library by the MARAS method ( Non-disease specimens) aptamer method. Through the design and implementation of positive screening and negative screening, the obtained aptamers only bind to certain disease-related biomolecules in positive samples, but not to non-disease-related organisms in positive and negative samples The molecules are combined, and then aptamers with the ability to distinguish positive and negative samples are selected from the nucleic acid library. At the same time, in order to verify the aptamer screening procedure, the obtained aptamers are used as detection probes to perform reverse verification on the positive samples and negative samples used in the screening to verify the correctness of the screening process. Moreover, in order to test the applicability of the aptamers screened for clinical testing, the obtained aptamers are used as detection probes for some blind test samples (non-positive or (Negative specimen) Perform blind sample analysis and compare with known pathological test results, and then check the ability of the obtained aptamer to distinguish between positive and negative blind test samples.

Other aspects of the present invention relate to using the obtained aptamers with the ability to distinguish between positive and negative specimens as capture ligands in purification reagents, and using virtual filters simulated using the oscillating magnetic field range to purify from positive attribute samples Some biomolecules related to disease, and the identification of the purified sample to identify the identity of the biomolecules purified, and then confirm the biomarkers related to disease. Because the identity of the biomolecules purified during the purification process cannot be known before purification, the binding affinity (or equilibrium dissociation constant) between these biomolecules and the obtained aptamers cannot be measured in advance, and the magnetic properties of the biomolecules when purified The structure of the particle-binding complex (target-aptamer-magnetic particle) is different from that of the screening (aptamer-target-magnetic particle), so the induced effect on the aptamer-biomolecule binding pair under the same oscillating magnetic field conditions The pulling force varies depending on the composition of the outermost layer of the binding complex (target relative to aptamer), so the oscillating magnetic field conditions that should be used for purification cannot be known before purification. However, the binding affinity between the aptamer obtained by MARAS screening and the target biomolecule is a function of the conditions of the applied oscillating magnetic field, and the greater the frequency and/or strength of the magnetic field, the greater the frequency between the obtained aptamer and the target biomolecule. The higher the binding affinity (the lower the equilibrium dissociation constant), MARAS provides a mechanism for the selection of the binding affinity between the aptamer and the target biomolecule. The technique disclosed in another aspect of the present invention is to purify unknown biomolecules from samples based on the characteristics of MARAS's binding affinity selection ability between the aptamer and the target biomolecule, using the manipulation of the frequency/intensity of the oscillating magnetic field The range is to select different biomolecules with different binding affinity ranges from the positive attribute samples and the obtained aptamers, and then purify some disease-related biomolecules from the positive attribute samples.

Before demonstrating the purification of certain disease-related biomolecules from disease patient specimens (positive attribute samples), it is necessary to screen out from the nucleic acid library samples with distinguished disease patients (positive screening samples) and non-disease patient tests (negative screening samples) ) Is used as a capture ligand for purification. In the present invention, the aptamers obtained by MARAS are further used as capture ligands to purify certain disease-related biomolecules from disease patient samples (positive attribute samples) to verify the feasibility of the present invention. As shown in FIG. 1, a procedure for selecting (or generating) an aptamer having the above-mentioned characteristics is schematically illustrated. The procedure depicted in Figure 1 is about four main parts: (1) material preparation; (2) negative selection; (3) positive selection; and (4) post-analysis, including PCR (polymerase chain reaction) amplification , Cloning, sequencing, reverse verification, and blind sample analysis. The method steps or details of the four main parts are described below.

In the process of material preparation, J non-disease patient specimens were prepared as negative screening samples (negative samples), and 1 disease patient specimens were prepared as positive screening samples (positive samples), and K specimens ( Including disease patients and non-disease patients) as samples for blind analysis (blind test samples). Individually biotinylation (biotinylation) and then combined with streptavidin (streptavidin) coated magnetic particles (SA-MPs) to form negative samples-magnetic particles (NS-MPs _(j) ), positive samples -Magnetic particles (PS-MPs _(i) ) and blind test samples-Magnetic particles (BS-MPs _(k) ). The subscripts j, i, and k of NS-MPs, PS-MPs, and BS-MPs are independently positive integers from 1 to J, I, and K, respectively. It should be noted that in the entire examples of reverse verification, blind sample analysis, and biomolecule purification using aptamers and aptamers, the magnetic particles (MPs) used are not limited to magnetic nanoparticles (MNPs) and are magnetic The use of microparticles (MMPs) in the following experiments will achieve similar results as the implementation in the MARAS program. The oligonucleotide library contains randomized oligonucleotide sequences flanked by primers at both ends for PCR amplification. A set of primers labeled Lab-forward primer and Lab-reverse primer are used to anneal the degenerating region of the resulting oligonucleotide (aptamer) during PCR amplification . Universal T7 primers are used to sequence the nucleotides of the selected aptamers.

For negative selection, first dissolve the nucleic acid library in phosphate buffer (Phosphate Buffer Saline, PBS, pH 7.4), then increase the temperature to 95°C (5 minutes), then quickly cool to 4°C (2 minutes) ), and then placed at room temperature (60 minutes) to make the nucleic acid form a specific secondary or tertiary structure, the nucleic acid library is incubated with NS-MPs ₍₁₎ . After incubation, magnetic separation was performed to remove the binding mixture and collect the supernatant containing the remaining oligonucleotides that were not bound to NS-MPs ₍₁₎ . Next, the collected supernatant was incubated with the next NS-MPs ₍₂₎ , and the process was repeated until the negative selection of all J NS-MPs _{(j) was} completed. The purpose of multiple rounds of negative selection operations is to consider that the composition of individual negative samples is not the same, so that the possibility of binding between the aptamers obtained by screening and the biomolecules in all J negative samples is minimized to increase The ability of aptamers to distinguish samples from disease patients. The same principle can be applied to the screening of aptamers for immunoassay to enhance the detection sensitivity. The aptamers screened in immunoassay can only be combined with the target analyte in order to reduce the possibility of false positive detection during the application of immunoassay. Theoretically, the more negative selection operations are performed (the greater the J), the higher the sensitivity that can be achieved during immunoassay (less false detections), and the higher the discrimination rate for disease patients and non-disease samples . The final supernatant after the negative selection was collected was used for the following positive selection. Alternatively, a single negative selection operation from all NS-MPs _(j) mixed NS-MPs instead of multiple rounds of negative selection operations using individual NS-MPs _(j) will achieve the same result. However, it must be noted that with mixed NS-MPs, the concentration of MPs in the PBS buffer may become too high, making the MPs easy to coalesce and adversely affect the screening. On the other hand, in biological systems, specific biomolecules may be present in the specimens of diseased patients and non-disease patients, but only in different concentrations. If the concentration of the specific biomolecule contained in the specimen of the non-disease patient is much lower than that contained in the sample of the diseased patient, the specific biomolecule can also play the role of a biomarker for diagnosing the disease. In this case, because the negative sample contains these specific biomolecules, the negative screening process in the aptamer screening will exclude some of the nucleic acids that may be bound to these specific biomolecules, thereby reducing the screening efficiency, but due to these specific organisms in the negative sample The concentration of molecules is very low and the total amount of nucleic acids contained in the used nucleic acid library is large. The negative screen designed by the present invention does not affect the output of the final screened aptamers.

For positive selection, the supernatant collected from negative selection is incubated with PS-MPs ₍₁₎ in PBS buffer. After incubation, magnetic separation was performed to collect the binding mixture and disperse the binding mixture in PBS buffer, while discarding the supernatant containing unbound oligonucleotide. The mixture was subjected to a binding having a lower cutoff frequency f _L and / or lower the off MARAS first intensity oscillating magnetic field H _L, moiety bound to PS-MPs ₍₁₎ is smaller than the affinity of a first oscillating magnetic field induced by the bonding of the The pulling force nucleic acid is detached from the binding mixture, and the supernatant containing the detached nucleic acid is removed by magnetic separation, and the collected binding mixture is re-dispersed in PBS buffer. Next, a second oscillating magnetic field having an upper limit cut-off frequency f _U and/or an upper limit cut-off intensity H _U is applied to the binding mixture solution to separate nucleic acids (aptamers) that bind to PS-MPs ₍₁₎ with the desired binding affinity, wherein The required binding affinity is greater than the pulling force induced by the first oscillating magnetic field on the bonding pair between the aptamer and the biomolecule and less than the pulling force induced by the second oscillating magnetic field on the bonding pair. Magnetic separation is performed to remove the binding mixture and collect the supernatant containing aptamers with the desired binding affinity range for PS-MPs ₍₁₎ . Next, the supernatant was incubated with the next positive sample (PS-MPs ₍₂₎ ), and the procedure was repeated until the positive selection of all I PS-MPs _{(i) was} completed. The purpose of multiple rounds of positive selection operations is to consider the differences in the composition of individual positive samples, so that the aptamers obtained can only be combined with biomolecules shared by all I positive samples, so as to increase the aptamers to disease The ability of the patient to distinguish the specimen, thereby reducing false negatives at the time of discrimination. Then, after completing the positive selection operation, the supernatant containing the aptamer is collected for post-analysis, such as PCR amplification, cloning, sequencing, reverse verification, and blind sample analysis for blind test samples. FIG. 1 schematically illustrates a procedure for generating an aptamer having a disease patient sample (positive screening sample) and a non-disease patient test (negative screening sample), including negative and positive selection. To achieve the above purpose, during the application of the oscillating magnetic field, constraints are imposed on the frequency (f) and the magnetic field strength (H) of the applied magnetic field, that is, f _L ≤ f _U and/or H _L ≤ H _U , and the equation cannot They exist at the same time, which means that f _L = f _U and H _L = H _U cannot be used at the same time. The oscillating magnetic field used can be a rotating, alternating or elliptical magnetic field. In addition, it is worth mentioning that for each round of positive screening, the oscillating magnetic fields used (f _L , H _L , f _U and H _U ) are not strictly required and only need to provide the aptamers obtained by screening The scope of the oscillating magnetic field with high binding affinity is sufficient, and the above-mentioned Chinese invention patent (ZL 2014 1 0570602.0) provides a reference for selecting the oscillating magnetic field range according to the required binding affinity (equilibrium dissociation constant) range, and also provides the Details of the execution procedures for PCR amplification, cloning and sequencing. Alternatively, positive selection in each round operation, is applied and having a lower cut-off frequency f or the _L / H _L lower cut-off a first intensity oscillating magnetic field, so that the collected redispersed binding mixture, heating, eluted, and purified by magnetic separation To obtain the supernatant containing the aptamer for the next round of positive screening or post-analysis, and the binding affinity of the obtained aptamer to the biomolecule binding pair is greater than that induced by the first oscillating magnetic field The pulling force of the bonding pair between body and biomolecule. Next, the screened aptamers are subjected to PCR amplification, cloning and sequencing, and these aptamers are used as detection probes to perform reverse verification on negative screening samples and positive screening samples. In addition, as in the MARAS program, the negative screening is performed before the positive screening or the positive screening is performed before the negative screening. The screening results for the aptamers are similar. In the technology disclosed in the present invention, the order of positive and negative screening It does not affect the results of screening aptamers. Once the screened aptamers have been verified (reverse verification), they can be used as detection probes in immunoassays to perform blind analysis or to purify reagent capture ligands to purify disease-related samples from samples with positive attributes Specific biomarkers.

It is also worth mentioning that if the composition of the negative sample is fixed, only one sample for negative screening is required (J = 1), and if the target biomolecule for detection or purification is fixed, only one sample for positive screening is required (I = 1 ). For example, when the former wants to identify or purify metabolites related to specific tumor cells from the tumor cell culture fluid, the normal cell culture fluid is used as a negative sample. Because the culture fluid is produced in a controlled environment and its components are fixed, it only needs to be Single negative screening sample. The latter is used to screen aptamers with high specificity and binding affinity for specific target biomolecules (targets) for use as detection probes in immunoassays or to use screened aptamers for purification from samples. When the target biomolecule of known identity is used as the capture ligand, because the target or target biomolecule is fixed and known, only the known target or target biomolecule needs to be used as a positive sample for positive screening, so only a single positive screening is required sample.

In addition, the competitive mechanism for aptamer selection provided by MARAS is based on the application of pulling force on the bonding pair between the aptamer and the biomolecule, so any method or facility that can produce a pulling force on the bonding pair can be used similar to MARAS Provide a competitive mechanism for aptamer screening, including (but not limited to) mechanical forces (such as the combination of magnetic particles and aptamer-biomolecules in aqueous solution, motion driven by an oscillating magnetic field; in aptamer-biomolecules Of the binding complex attached to a fixed substrate or a shut-off (eg fluid channel) induced by rigorous washing; and in a disk laboratory (lab-on-a-disc) by attaching to the fixed Centrifugal force induced by the rotation of the aptamer-biomolecular complex of the substrate or capture (eg fluid channel); electromagnetic force (eg static magnetic force induced by a gradient magnetic field in the case of using magnetic substances and using charged substances In the case of static electricity induced by an electric field); or any combination of the above forces.

The reverse verification and blind sample analysis can be performed by techniques commonly used in general medical tests such as enzyme-linked immunosorbent assay (ELISA), real-time quantitative PCR (q-PCR), or nephelometry. In the following, the present invention will briefly describe the steps of reverse verification and blind sample analysis by using the obtained aptamers as detection probes by q-PCR method. First, the excess aptamer is heated in PBS buffer and rapidly cooled as described above to form a secondary or tertiary structure, and then the sample-magnetic particles (J NS-MPs _(j) and I PS -MPs _(i) ) and blind-test sample-magnetic particles (K BS-MPs _(k) ), then perform magnetic separation to remove the supernatant containing unbound aptamers and retain the magnetic particle binding complex, Wash with PBS buffer to completely eliminate non-specific binding, and then disperse and heat the magnetic particle binding complex with deionized water to elute the aptamer bound to the magnetic particle binding complex for magnetic separation. The magnetic particles were removed and the supernatant containing aptamers was retained. Finally, the amount of aptamers contained in the supernatant was measured by q-PCR and presented in relative performance. It is worth mentioning that in the blind sample analysis, in order to enhance the possibility of non-specific binding to a minimum, the first oscillating magnetic field conditions (with a lower cut-off frequency f _L and / Or the lower limit cut-off strength _HL of the oscillating magnetic field) to detach the aptamer from which the binding affinity between the aptamer and the biomolecule is less than the pulling force on the bonding pair between the aptamer and the biomolecule induced by the first oscillating magnetic field And through magnetic separation and exclusion to further exclude non-specific binding.

In the present invention, the purification step of using the aptamer obtained by the foregoing procedure as a capture ligand to purify certain disease-related biomolecules from a disease patient specimen (positive attribute sample) is to use an oscillating magnetic field to bind Magnetic particles with aptamers as ligand-capturing ligands combine with certain specific biomolecules associated with diseases to generate magnetic particle binding complexes to produce a pulling force on the bonding pair between aptamers and biomolecules, which in turn causes the magnetic particles The specific biomolecule with a specific binding affinity range in the binding complex is detached from the magnetic particle binding complex, and then the supernatant is collected through magnetic separation to obtain the specific biomolecule with a specific binding affinity range for the aptamer. Because the structure of the magnetic particle binding complex (target-aptamer-magnetic particle) and the structure of the screening (aptamer-target-magnetic particle) are different during the purification process, the induced effect on the aptamer and The pulling force of the bonding pair of biomolecules (defined by the viscous force generated when the magnetic particle binding complex moves in the liquid in the opposite direction of the movement), because the composition of the outermost layer of the binding complex is different (target relative to aptamer) There are differences, so the oscillating magnetic field conditions that should be used for purification cannot be known before purification. Theoretically, the larger the outermost component, the greater the pulling force induced by the same oscillating magnetic field on the bonding pair between the aptamer and the biomolecule. Generally speaking, the size of the biomolecule to be purified is larger than that of the aptamer, so the same The pulling force induced by the oscillating magnetic field on the coupling pair between the aptamer and the biomolecule during purification is greater than the pulling force induced during the screening, on the other hand, the binding affinity of the aptamer and the biomolecule to be purified is fixed, thus It is expected that the oscillating magnetic field conditions required to remove the biomolecule to be purified from the aptamer during purification are less than the oscillating magnetic field conditions required to remove the aptamer from the biomolecule during screening. Therefore, in the present invention, the scanning of the oscillating magnetic field conditions, that is, the scanning method of binding affinity is performed to obtain and purify disease-associated biomolecules with different binding affinity ranges of the aptamer. On the other hand, because the amount of samples (positive attribute samples) of patients with a single disease may be limited, the amount of biomolecules purified according to the above steps is not enough to be sequenced by a mass spectrometer to confirm identity, so the same oscillating magnetic field conditions Specimens (different positive attribute samples) of patients with different diseases of the same disease are scanned for binding affinity range, and biomolecules with the same binding affinity range as the aptamer are collected and collected for mass spectrometry analysis and identification.

Preparation of purification reagents must be carried out before purification. Regarding the preparation of purification reagents, the aforementioned validated aptamers are first used as capture ligands for purification reagents, and excess biotinylated aptamers are used in PBS buffer After heating and rapid cooling to form a secondary or tertiary structure as described above, incubation with SA-MPs, magnetic separation to remove the supernatant containing aptamers not bound to the magnetic particles and retain the magnetic particle binding complex PBS buffer to disperse the magnetic particle binding complex as a purification reagent. After the purification reagent is prepared, it can be used to purify a blind test sample that has a high relative amount of positive properties when the aptamer is subjected to blind sample analysis by q-PCR. The flow chart of the purification of unknown biomolecules related to disease in the sample is shown in Figure 2.

Fig. 2 illustrates that firstly, N samples (positive attribute samples) with high relative performance of the aptamer are selected as the samples to be purified by the q-PCR results of the blind sample analysis, and then the positive attribute samples (1) Incubate with the purification reagent, magnetically separate the supernatant containing the part that is not bound to the magnetic particles with aptamers in the purification reagent and collect the magnetic particle binding complex, and then wash with PBS buffer several times to disperse the magnetic The particle-bound composite, followed by applying an initial (zeroth) oscillating magnetic field having a magnetic field frequency f ₀ and a magnetic field strength H _{0 to} the dispersion solution of the magnetic particle-bound composite, so that the magnetic particle-bound composite is lower than the initial (zeroth) ) The non-specific binding affinity or the partial binding of the biomolecule bound to the aptamer with low binding affinity is detached, and the supernatant containing the detached portion is removed by magnetic separation again, and the magnetic particle binding complex is collected and washed with PBS buffer Several times and dispersed, where the initial binding affinity is defined by the pulling force induced by the initial oscillating magnetic field for the binding pair between the aptamer and the biomolecule. The first oscillating magnetic field having the magnetic field frequency f ₁ and/or the magnetic field strength H ₁ is applied to the dispersion solution of the magnetic particle binding complex again so that the binding affinity of the magnetic particle binding complex and the aptamer is between the initial binding affinity Partial separation of biomolecules with the first binding affinity, magnetic separation to collect the supernatant containing the separated part (separation component (1)), while dispersing the magnetic particle binding complex with PBS buffer, and then applying a magnetic field A second oscillating magnetic field of frequency f ₂ and/or magnetic field strength H ₂ , and repeatedly perform the purification procedure for the positive attribute sample (1) until the purification and collection of all M separated components is completed, wherein the first binding affinity system The pulling force induced by the first oscillating magnetic field for the binding pair between the aptamer and the biomolecule is defined, and the binding affinity range defined by the initial and first oscillating magnetic field can be regarded as a virtual filter (1) To select biomolecules with binding affinity between aptamers within the above range. In other words, for the purification process of unknown biomolecules of any positive attribute sample (n), a total of M rounds are performed, that is, M virtual filters are used. After the final round of purification of the positive attribute sample (1) and collection of the separated component (M), the magnetic particle binding complex is dispersed in PBS buffer and used as the purification reagent for the next positive attribute sample or according to the preparation of the above purification reagent Method to prepare purified reagents again. Next, the positive attribute sample (2) and the purification reagent are incubated together to purify the next positive attribute sample (2), and the purification procedure for the positive attribute sample (n) is repeated until all N positive attribute samples are completed Purification and collection of separated components. According to the above purification procedure, all the collected biomolecules in the separated component (m), whose binding affinity with the aptamer as the capture ligand ranges from the (m-1)th applied oscillating magnetic field ( f _m-1 , H _m-1 ) and the (m) oscillating magnetic field (f _m , H _m ) induce a pulling force between the pair of aptamer and biomolecule. This means that by applying an oscillating magnetic field to induce a pulling force on the binding pair between the aptamer and the biomolecule, the biomolecule with a certain range of binding affinity to the aptamer is filtered from the positive attribute sample, and then the Biomolecules with a specific binding affinity purified from samples with positive attributes can be regarded as virtual filters. In addition, as described above, the oscillating magnetic field conditions required to remove the biomolecules to be purified from the aptamers during purification are less than the oscillating magnetic field conditions to remove the aptamers from the biomolecules during screening, so the highest used in the purification process The conditions of the oscillating magnetic field (the (M) oscillating magnetic field: f _M and H _M ) can be defined by the second oscillating magnetic field (f _U and H _U ) at the time of screening, and the final collected magnetic particles bind the complex due to all biological molecules All have been detached from the magnetic particle binding complex, so the collected remaining magnetic particle binding complex does not contain any biomolecules, so it can be provided as a purification reagent for the next positive attribute sample. Furthermore, in order to achieve the function of this virtual filter, the applied oscillating magnetic field must meet the following constraints: f _m-1 ≤ f _m and/or H _m-1 ≤ H _m , and the equation of frequency and intensity cannot It applies simultaneously, for example, f _m = f _m-1 and H _m = H _m-1 cannot be applied at the same time. In addition, in order to make the individual separation component (m) isolated from all N positive attribute samples and the aptamer as the capture ligand have the same binding affinity range, the purification of all N positive attribute samples The oscillating magnetic field conditions of the virtual filter used in the process must be consistent.

It is worth noting here that during the separation process only the mechanical force (pulling force) induced by the oscillating magnetic field is applied, and this force is very weak (if estimated by the force of specific bonding, it is about piconton level), and Without any influence of temperature and chemistry, the activity of the purified biomolecule will not be reduced due to the purification process. The biomolecule purified by this method can retain the activity of the biomolecule. This virtual filter can be used in the purification of unknown or known biomolecules. The former is used as a ligand-capturing aptamer in the purification and the aptamers obtained by the screening are used to purify the unknown from the sample. Identity biomolecules can be carried out by the method of the present invention. The latter is relatively simple, because the identity of the biomolecule is known at the time of aptamer screening, so it is only necessary to use this biomolecule as a sample for positive screening, and a round of positive screening can be used to obtain the detection probe and It is used as an aptamer to capture ligands during purification, and then purified by the virtual filter disclosed in the present invention. If a biomolecule has been purified once from a sample, the range of the oscillating magnetic field (the (m-1) magnetic field and the (m) magnetic field) applied during the purification of the biomolecule is known, and the same for subsequent When the aptamer is used as the capture ligand to purify the specific biomolecule from the sample, only the known oscillating magnetic field range needs to be applied, and the oscillating magnetic field scan is not required, that is, only one virtual filter is needed. No M virtual filters are required.

It is also worth mentioning that the virtual filter designed according to the binding affinity range of the bonding pair between the aptamer and the biomolecule is based on the mechanism of applying a pulling force to the bonding pair, so any one that can produce a pulling force on the bonding pair Methods or facilities can be used to generate a mechanism similar to the oscillating magnetic field described above as a virtual filter, including (but not limited to) mechanical forces (such as the binding complex of magnetic particles and aptamer-biomolecules in an aqueous solution, via Pulling force induced by motion driven by an oscillating magnetic field; fluid dynamics induced by rigorous washing with aptamer-biomolecular complex attached to a fixed substrate or entrapped (eg, fluid channel); and in a disk laboratory (lab-on-a-disc) centrifugal force induced by the rotation of an aptamer-biomolecular complex attached to a fixed substrate or captured (e.g. fluid channel), etc.; electromagnetic force (e.g. In the case of matter, the static magnetic force induced by the gradient magnetic field and the static electricity induced by the electric field in the case of using a charged substance); or any combination of the above forces. In order to implement a virtual filter mechanism based on the selected binding affinity range, the range of the pulling force generated by changing the oscillating magnetic field, the range of hydrodynamic force generated by changing the buffer washing speed in the fluid channel, and the The range of centrifugal force generated by rotation at different rotation rates and the range of electromagnetic force generated by changing the strength of the gradient magnetic field and/or electric field. It should be noted that the selection mechanism of the binding affinity range can be used to select aptamers with a desired binding affinity range during the aptamer selection process and can also be used to filter out the binding affinity to the aptamer during the biomolecule purification process Biomolecules in a specific range.

After completing the purification of all selected positive attribute samples with a virtual filter and collecting a total of M separated components of N positive attribute samples, the corresponding separated components (collected under the same magnetic field range) of all positive attribute samples are collected. Separated molecules) are concentrated for subsequent identification of the biomolecules contained in the collected separated components. Because the total solution of the M separated components collected contains PBS buffer and purified biomolecules, although the total amount of biomolecules is sufficient for the analysis of the mass spectrometer (it can be determined by the number of positive attributes samples N), but its The concentration is very low, so first concentrate, and separate the concentrated M separated components by polyacrylamide gel electrophoresis, observe the distribution of biomolecule bands on the film, and finally cut out the desired biomolecule bands and perform mass spectrometry Analysis to sequence the purified biomolecules.

The biomolecule obtained through the purification process disclosed in the present invention is a biomolecule related to a specific disease, and can be used as a biomarker for the disease. In reality, multiple aptamers may specifically bind to the same biomolecule, or a aptamer may specifically bind to multiple biomolecules but have different binding affinity. The condition of the former does not have any impact on the results of purification and identification, while the latter because of the virtual filter used in purification can separate biomolecules with different binding affinity for aptamers, so it will not affect the purification and identification. The result has a negative impact. However, a situation that may have a negative impact on the purification results may also occur. An aptamer can bind to multiple biomolecules and have the same binding affinity (for example, different biomolecules have the same part that fits the aptamer) At the same time, the size of the biomolecules is the same (it is not possible to separate different types of biomolecules by polyacrylamide gel electrophoresis). In this case, this purification procedure cannot separate different types of biomolecules, and different aptamers must be replaced. purification. Considering the above situation, in practice, multiple aptamers can be selected during aptamer screening, and the purification and mass spectrometry analysis disclosed by the present invention are repeated for positive attribute samples respectively to confirm multiple possible biomarkers for the disease Thing.

Example: Purification and identification of disease-related biomarkers ─ Take epidermal growth factor receptor mutant non-small cell lung cancer as an example

According to the biomarker discovery process of the specific disease disclosed by the present invention. It mainly includes a screening program for screening aptamers with the ability to distinguish specimens from disease patients and non-disease patients from the nucleic acid library, and purification of biomolecules with unknown identities of positive attribute samples using the obtained aptamers as capture ligands. The program and other two parts are shown in Figure 1 and Figure 2, respectively. In the following, unknown biomarkers related to disease will be purified and identified from serum samples of non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations as Examples to illustrate the specific content of the present invention. For non-small cell lung cancer, the EGFR mutation is located in the four exons (exon 18-21) of the kinase domain, which is concentrated in exons 19 and 21, accounting for the majority of patients with disease, including the 746- The 752 amino acid codon nucleotide deletion mutation (exon 19 deletion mutation: 45%) and the 858th amino acid codon have a TG conversion missense mutation (exon 21 missense mutation: 40%). This example takes EGFR-mutated NSCLC patients (including EXON 19 and L-858R) as an example. The main purpose is to explain in detail the technology disclosed in this patent application, including aptamer selection procedures and biomolecule purification procedures, rather than It is to present the discovery and definition of biomarkers for NSCLC with EGFR mutation diseases, so only one screened aptamer was used to demonstrate the purification procedure, and multiple aptamers were not selected to purify other possibilities from patient serum As a biomolecule for the biomarkers of the above diseases, at the same time only select a few purified sample ribbons on the electrophoresis film without scanning all the sample ribbons on the electrophoresis film with a mass spectrometer to confirm the identity of all purified samples, and no further Corresponding monoclonal antibodies are used to verify the correctness of these biomolecules as biomarkers for the above diseases.

Experimental setting of MARAS platform

The following describes the experimental setup for performing a method for screening aptamers with the ability to distinguish between positive and negative specimens on the MARAS platform and a method for purifying biomolecules from positive attribute specimens with the aptamers obtained by screening. . The experimental setup contains at least two sets of coils 100, power amplifier 200, signal generator 300 for generating an oscillating magnetic field and is operated with the LABVIEW computer program. The oscillating magnetic field used for MARAS may be a rotating magnetic field in the case of a rotating magnetic field-MARAS (RO-MARAS), or an alternating magnetic field in the case of an alternating magnetic field-MARAS (AC-MARAS). For RO-MARAS, a rotating magnetic field is generated by two sets of Helmholtz coils 100 placed vertically. The LABVIEW program is used to send two signals cos(ωt) and sin(ωt) to the dual-channel power amplifier 200 through the signal generator 300 and the NI BNC-2110 capture box. Then the two signals are amplified equally to drive two sets of coils simultaneously to generate a rotating magnetic field. The experimental setup is schematically shown in Figure 3A. In the figure, the sample is placed at the intersection of the center lines of the two groups of Helmholtz coils 100. However, other alternative arrangements can also be used to generate a rotating magnetic field. For AC-MARAS, an alternating magnetic field is generated by a single excitation solenoid 100 driven by a signal generator 300 and a current generator unit 200, as schematically shown in FIG. 3B, where the sample is placed Within the solenoid 100. It should be noted that if the computer program only sends a signal to a group of Helmholtz coils to generate an AC magnetic field, then the setup of FIG. 3A can also be used for AC-MARAS. In addition, other alternative settings can also be used to generate the AC magnetic field. In addition, other types of oscillating magnetic fields are also applicable, such as elliptical magnetic fields, which can be generated using the settings depicted in FIG. 3A by using different amplification factors of the power amplifier 200 for sine and cosine signals from the signal generator 300. In this example, the RO-MARAS platform is used to apply a rotating magnetic field, which is limited by the specifications of the power amplifier used. In the aptamer screening and biomolecule purification procedures, all the applied rotating magnetic fields are fixed at 14 gauss . Moreover, because the magnetic particles have biological molecules on the surface, they are easily combined with each other under the action of the magnetic field to make them aggregate into larger molecular clusters, and precipitation occurs. Therefore, in the screening and purification of the oscillating magnetic field, the particles are absorbed every 2.5 minutes. The measuring tube breaks up the deposited clusters of magnetic particles.

Material preparation

Before screening the aptamers disclosed in the present invention on the MARAS platform, materials need to be prepared. Material preparation includes preparing random oligonucleotide libraries and primers, preparing positive screening samples, negative screening samples, and blind testing samples. These preparation steps are outlined in the following sections.

Oligonucleotide libraries and primers

The initial oligonucleotide pool is 60-mers in length and consists of a randomized 20-mer middle segment (N20) and two primers with 20-mer fixed segments at both ends. The oligonucleotide sequence synthesized by chemical solid phase and purified by PolyAcrylamide Gel Electrophoresis (PAGE) is (5ꞌ-AGCAGCACAGAGGTCAGATG-N20-CCTATGCGTGCTACCGTGAA-3ꞌ (SEQ ID NO: 1)). A set of primers (labeled forward Lab-F: 5ꞌ-AGCAGCACAGAGGTCAGATG-3ꞌ (SEQ ID NO: 2) and labelled reverse Lab-R: 5ꞌ-TTCACGGTAGCACGCATAGG-3ꞌ (SEQ ID NO: 3)) were used for PCR amplification During the annealing of the 5'and 3'degenerate regions of the oligonucleotide library. The 5'-biotinylated primers Lab-Biotin-F and Lab-Biotin-R with the same sequence as above are used to take biotin-forward single-stranded and forward single-stranded nucleotides from double-stranded PCR products, respectively Separate. The universal T7 (T7: 5'-TAATACGACTCACTATAGGG-3' (SEQ ID NO: 4)) primer was used to sequence the nucleotides of the selected aptamers. All nucleic acid containing primers were purchased from Shengong (MDBio, Taiwan). It is worth noting that random oligonucleotide libraries of different lengths and sequences and their corresponding primers can be used without changing the results of the present invention.

Preparation of Serum Protein Nanomagnetic Bead Reagent for Biotin Calibration

The experiment of this example used five positive screening groups (I=5) as sera of NSCLC patients with EGFR mutations (including EXON 19(2) and L-858R(3)); another five negative screening groups (J=5) ) Is the serum of non-EGFR mutant NSCLC patients; in addition, 18 and 20 NSCLC patient sera that have been confirmed to be non-EGFR mutations and EGFR mutations by slice pathological analysis were used as blind test samples (K=38) for blind sampling analysis. The clinical data of the serum samples of NSCLC patients used are shown in Tables 1 and 2:

Table 1 Negative and positive screening serum clinical data of NSCLC patients

Table 2 Blind sample analysis of the clinical data of patients with NSCLC

Separately, the individual clinical serum samples in Tables 1 and 2 above were set with protein quantification kit (Bio-Rad protein assay, Bio-Rad, Taiwan) with enzyme immunoassay analyzer (ELISA Reader, Molecular Devices, Taiwan) at absorbance value 570 Determine the amount of protein in nm, and control the total protein amount to 100 μg, and then use the biotin calibration kit (EZ-Link™ Sulfo-NHS-Biotinylation kit, Thermo Scientific; Nanosep ® Centrifugal Devices, Life Science, USA) According to the manufacturer's instructions, biotin is calibrated on the protein of individual clinical serum samples, and then the biotinylated magnetic nanoparticle reagent coated with the biotinylated serum sample and the streptavidin-coated serum nanoparticle reagent ( SA-MNP reagent) 50 μL reacted at 4°C and mixed well for more than 16 hours to prepare magnetic nanoparticle reagent (sample reagent) for biotin-calibrated serum protein, which contains 5 positive screening samples and 5 negative Reagents for screening samples and 38 blind test samples (PS-MNP, NS-MNP, and BS-MNP) were stored in a refrigerator at 4°C until needed for the experiment. The SA-MNPs were dispersed in PBS buffer to form SA-MNP reagents, and were purchased from Magnetobiology (Magqu, Taiwan). The average hydrodynamic diameter of SA-MNPs in the reagent is 50 nm and the SA-MNP concentration of 0.3 emu/g. Before using SA-MNPs and sample-MNPs (including PS-MNPs, NS-MNPs, and BS-MNPs), these sample reagents must be magnetically separated and washed with PBS buffer at least twice before the subsequent experiments.

RO-MARAS method for screening aptamers with ability to distinguish specimens of disease patients

A single-stranded nucleic acid library with a quantity of 1 nM was dissolved in 100 μL of PBS buffer, and then the temperature was rapidly reduced after being heated as described above to make the nucleic acid form a specific secondary or tertiary configuration. After completion, the five sets of negative screening sample reagents prepared above were washed twice with PBS buffer and magnetically separated to obtain NS-MNPs, and the NS-MNPs ₍₁₎ and the single-stranded nucleic acid library were placed in a rotary mixer Mix thoroughly for 1 hour, then use a strong magnet to perform magnetic separation, retain the supernatant, remove the nucleic acids that will bind to any protein in all NS-MNPs ₍₁₎ , and then remove the supernatant and the remaining nucleic acids. The next group of negative screening samples NS-MNPs ₍₂₎ are incubated together, repeat the above step J (5) round, and complete the negative screening of the remaining NS-MNPs ₍₂₎ to NS-MNPs ₍₅₎ in sequence, and then take out the last The supernatant was purified with nucleic acid purification kit (Gel/PCR DNA Isolation System, Viogene, Taiwan) and dissolved in 100 μL of PBS buffer and collected, and the negative screening was completed.

Next, enter the positive screening, and the nucleic acid-containing supernatant that has been negatively screened and purified is firstly heated up as described above and then quickly cooled down, so that the nucleic acid forms a specific secondary or tertiary configuration, and the five positive prepared above are separately prepared. Screening reagents were washed twice with PBS buffer and separated magnetically to obtain PS-MNPs. After that, the supernatant of the nucleic acid library after negative screening was incubated with the positive screening sample-magnetic particles (PS-MNPs ₍₁₎ ) of EGFR-mutated NSCLC patients, and placed on a rotary mixer to mix thoroughly for 1 hour, and removed by magnetic separation After the supernatant, the PS-MNPs _{(1) are} bound to the nucleic acid-binding complex and washed with PBS buffer several times and dissolved back (100 μL), and then under specific rotating magnetic field conditions (magnetic field frequency: 27 KHz, magnetic field strength: 14 gauss) MARAS screening, rejection of weakly binding nucleic acids. The biological particles on the surface of the magnetic particles are easy to combine with each other to make them aggregate into larger molecular clusters, and precipitation occurs. Therefore, the deposited magnetic particle molecular clusters are dispersed by a pipette every 2.5 minutes for a total of 10 minutes. After that, magnetic separation is performed to remove the supernatant, leaving the PS-MNPs ₍₁₎ and nucleic acid binding complex and washed several times with PBS buffer. The remaining PS-MNPs ₍₁₎ and nucleic acid binding complex are the nucleic acids For the desired nucleic acid with strong binding affinity to the protein bound to PS-MNPs ₍₁₎ , the binding complex was dispersed with 20 μL of ddH ₂ O to disperse the cluster of magnetic particles and heated to 95°C for 10 minutes. To detach the nucleic acid, magnetically separate the supernatant containing the detached nucleic acid, and then use a nucleic acid purification kit to purify it and re-dissolve it in 100 μL of PBS buffer. After tertiary configuration, incubate with PS-MNPs ₍₂₎ , repeat the positive screening step I (5) above, and complete the positive screening of the remaining PS-MNPs ₍₂₎ to PS-MNPs ₍₅₎ in sequence, It can be obtained that it does not bind to all the proteins on NS-MNPs _(j) (j=1~5), but only binds to the proteins contained in PS-MNPs _(i) (i=1 ~5) and has high binding Affinity aptamers, or aptamers associated with a few diseases commonly contained in negative screening samples and at low concentrations, and aptamers commonly contained in positive screening samples and at relatively high concentrations of protein binding and having high binding affinity. The experimental process is shown in Figure 1. The circle on the right of the figure is the circle for negative screening. At this time, it is not necessary to go through MARAS, but to exclude single-stranded nucleic acids that will bind to the NS-MNPs of all 5 negative screening samples. Next, enter the positive selection loop on the left. At this time, the competitive mechanism provided by MARAS is used to keep the single-stranded nucleic acids with strong binding affinity on the PS-MNPs, and then use heating to separate them from the MNPs. Purification to facilitate follow-up experiments, and the use of five negative screening samples and five positive screening samples is mainly to improve the specificity and sensitivity of the selected aptamers, so as to reduce the false negative and false positive for subsequent detection And the trouble caused by purification.

Nucleotide aptamer sequence sequencing

The selected aptamers were amplified by PCR with Lab-F and Lab-R primers respectively. The PCR reaction contained 1.25 units of DNA polymerase (Invitrogen Life Technologies, Grand Island, NY, USA), 0.1 mM dNTPs, The 0.5 mM MgSO ₄ and 0.5 nM primers were performed under the following conditions: 10 minutes at 94°C; 40 seconds at 94°C, 40 seconds at 57°C, 40 seconds at 72°C, 35 cycles; 72°C Next 10 minutes. The PCR product is purified by using a nucleic acid purification kit. The purified product was subclone into pGEM-T Easy vector (Promega, Madison, WI, USA). The cloning procedure is performed according to the manufacturer's instructions. The randomly selected three colonies of plasmids (plasmids) were purified by using a high-speed plasmid small reagent kit (Geneaid, Taipei, Taiwan). Plasmids were obtained by using Applied Biosystems PRISM 3730 DNA automatic sequencer and Big Dye terminator cycle sequencing kit (Foster City, CA, USA) ) Sequencing. Using the MARAS method, three aptamers that can bind to NSCLC EGFR-mutated disease-associated biomolecules were selected. After PCR amplification and colonization, the sequencing results are shown in Table 3.

Table 3 Sequence of N20 region of aptamers screened by MARAS

Preparation of Screened Forward Single Strand Aptamers

In order to confirm whether the screened aptamers can actually have corresponding binding with the screening control group (positive screening samples and negative screening samples), PS-MNPs and NS-MNPs must be used in reverse with the screened aptamers Verification. Use the nucleic acid plasmids of the three aptamers in Table 3 as templates to perform PCR amplification using Lab-F/Lab-Biotin-R as primers (the reaction conditions are the same as above), and purify the amplified products using nucleic acids The kit was purified and re-dissolved in 100 μL of PBS buffer, and then the PCR product was reacted with SA-MNPs for 2 hours. The supernatant that was not bound to SA-MNPs was removed by magnetic separation and the magnetic nanoparticle binding complex was dispersed. In 100 μL PBS buffer, add freshly prepared 0.15 M NaOH and react for 4 minutes to break the double-stranded binding of nucleic acids. After magnetic separation, collect the supernatant and remove the magnetic particles with biotin-reverse single-stranded nucleic acid. The collected supernatant was added with 100% alcohol 1 mL, placed in a refrigerator at -80°C for 2 hours, then centrifuged at 12000 rpm for 15 minutes, the supernatant was removed, and then 75% alcohol 1 mL was added to Centrifuge at the same speed for 15 minutes to remove salts. After removing the supernatant, place it in a dry bath at 70°C to evaporate the residual water and alcohol. After drying, add 100 μL of PBS buffer to dissolve the precipitate to obtain Purified positive single-strand aptamer. After the single-stranded aptamer is purified, it must be first spectrophotometer (NanoDrop 2000c, Thermo Fisher Scientific) (Thermo Fisher Scientific, Wilmington, DE, USA) Determine the concentration, and then dilute the three aptamers with PBS buffer to a fixed concentration of 10 nM for later reverse verification and blind sample analysis.

Using q-PCR to evaluate the combination of aptamers screened by reverse verification and screening control groups

100 μL of the three aptamers (Aptamer37, Aptamer44 and Aptamer48) were diluted to a concentration of 10 nM, as described above, and then quickly cooled down to make the nucleic acid form a specific secondary or tertiary configuration. Five PS-MNPs _(i) (i =1~5) and five NS-MNPs _(j) (j = 1~5) were mixed thoroughly at room temperature for 60 minutes, the supernatant was removed by magnetic separation, and then The magnetic particle binding complex was washed several times with PBS buffer, after removing the unbound aptamer, finally disperse the magnetic particle binding complex with 10 μL ddH ₂ O, heat to 95°C and react for 10 minutes to destroy the aptamer and The bond between the magnetic particles, and the supernatant containing the aptamer is collected by magnetic separation.

Next, q-PCR (StepOne™ Real-time PCR system, Applied Biosystems) was used to measure the content of the aptamers collected in the supernatant containing the aptamers in duplicate, and the fluorescence emitted by each cycle was analyzed. Intensity, when the release intensity reaches the threshold (Threshold) preset by the system, the number of PCR cycles is calibrated as Ct (Threshold cycle), so the resulting Ct value is inversely proportional to the content of the initial aptamer. Each q-PCR reaction mixture is 10 μL, containing 5 μL of SYBR Green PCR master mix (Applied Biosystems), 0.5 μL of Lab-F/Lab-R (0.5 nM), and 4 μL of the previously collected supernatant sample. The reaction conditions are as follows: 5 minutes at 95°C; 40 seconds at 94°C, 40 seconds at 60°C, and 40 seconds at 72°C for 40 cycles; 94°C for 10 minutes. The formula for estimating the content of aptamers based on the Ct value is: nucleic acid relative expression = 2 ^-Ct .

In order to confirm whether the screened aptamers are indeed capable of identifying the sera of NSCLC patients with and without EGFR mutations, reverse verification was performed to confirm, and the results are shown in FIGS. 4A-4C. It can be found in the figure that the amount of binding of the three aptamers screened to the screening control group corresponds to their relative performance, and there are very significant differences in the screening samples of NSCLC patients with and without EGFR mutation, and then verified according to the figure 1 The flow chart reveals the correctness of the implementation process of aptamer screening.

The blind sample analysis of blind test samples by q-PCR method for the selected aptamers

Further, the three aptamers were blindly analyzed to detect whether there are unknown biomolecules that can be bound by the aptamers in the blind test sample, and the same was carried out in a duplicate manner of q-PCR method (the reaction conditions were the same as above). First, the aptamer with a fixed concentration of 100 μL (10 nM) is heated as described above and then quickly cooled down to form a specific secondary or tertiary structure, and then separately with each BS-MNPs _(k) (k = 1 ~38) After uniformly mixing the reaction at room temperature for 60 minutes, the supernatant was removed by magnetic separation, washed several times with PBS buffer, and then the binding complex of BS-MNPs was dispersed with 100 μL of PBS buffer, and a specific rotation was applied Magnetic field (magnetic field frequency: 27 KHz, magnetic field strength: 14 gauss), and disperse the deposited clusters of magnetic particles with a pipette every 2.5 minutes as described above, and react for a total of 10 minutes to allow non-specific or low binding The part of the affinity-bound aptamer was detached from the binding complex of BS-MNPs _(k) , and the supernatant containing the detached aptamer was removed by magnetic separation. Finally, 10 μL of ddH ₂ O was used to disperse the BS- The binding complex of MNPs was heated to 95°C for 10 minutes to break the bond between the aptamer and the magnetic particles, and the magnetic particles were removed by magnetic separation and the supernatant was collected. Next, the collected supernatant was analyzed by q-PCR in duplicates as described above to analyze the relative expression of each aptamer bound to the biomolecule in each BS-MNPs _(k) .

The aptamers (Aptamer37, Aptamer44, and Aptamer48) that have been screened and reverse-validated will be used for blind analysis of blind samples. That is, the BS-MNPs made from the sera of other NSCLC patients in the non-screening control group were blindly analyzed. They were also expressed in terms of their relative performance after q-PCR analysis, and were compared with the NSCLC patients in the blindly tested group after pathological analysis. Serum clinical data table for comparison, the results are shown in Figure 5A ~ Figure 5C. As a result, it can be found that each aptamer has different levels of binding to different blind test samples, indicating that the amount of each aptamer in the serum samples of different patients binds to molecules of unknown biological targets. In addition, it can also be found that the relative performance of the positive blind test samples is significantly higher than that of the negative blind test samples, and the positive blind test samples with high individual aptamer performance are not exactly the same. The former shows that the selected aptamers have the ability to distinguish specimens of NSCLC patients with and without EGFR mutation, while the latter shows that the biomolecules in the serum of positive patients bound by individual aptamers may be different. In addition, all aptamers also have lower and varying degrees of relative performance in the negative blind test samples than the positive blind test samples. This should be due to the presence of the biomolecules bound by the aptamers in the negative and positive In patient samples, the concentration in the serum of positive patients is much higher than that in the serum of negative patients. It is worth mentioning that, because the serum of some blind test samples has been used up, the aptamer has no data on the relative performance of the q-PCR combined with the blind test samples. In addition, because there is no complete medical records, the appropriate The relative performance of the ligand for certain positive blind test samples is low, possibly because the positive blind test samples were collected after the patient had undergone medical behavior. However, these two situations do not affect the interpretation of the results. Further, the relative performance of individual aptamers combined with BS-MNPs in blind analysis was used to compare the clinical data with age, gender, cancer stage, smoking, and EGFR mutations, using Mann-Whitney U Test Statistical analysis is available in Table 4. From the table, it can be clearly seen that the selected aptamers are significantly related to the presence or absence of EGFR mutations (p-value <0.05), but not so significant for other clinical data Of relevance. The results of statistical analysis further verify that the selected aptamers have the ability to distinguish specimens of NSCLC patients with and without EGFR mutation.

Table 4 Correlation between the amount of aptamers and blind test samples and clinical factors

Purification of unknown biomarkers in samples with positive attributes In this example, Aptamer37 aptamers were selected for demonstration as purification ligands that could be used as biomarkers for the purification of NSCLC with EGFR mutation diseases from blind samples of positive attributes and selected The relative performance of binding to Aptamer37 aptamer is high and there are more serum positive test samples (patient number: No. 22, 31, 177 and 396) as purified samples. The purification reagent must be prepared before purification. The preparation method of the reagent is as follows.

Preparation of Purified Reagents Using Screened Aptamer (Aptamer37) as Capture Ligand

Using the aptamer (Aptamer37) as a template, a PCR reaction was performed using Lab-Biotin-F/Lab-R, and the PCR reaction conditions were as described above. After the reaction, the PCR product was purified with a nucleic acid purification kit and re-dissolved in 100 μL of PBS buffer, followed by adding SA-MNPs to react for 2 hours. Since the PCR product is a double-stranded aptamer and is hybridized by forward and reverse single-strand It has biotin at its 5 forward end, so the double-stranded aptamer will bind to SA-MNPs. Magnetic separation removes the supernatant that is not bound to SA-MNPs and disperses the magnetic nanoparticle binding complex. In 100 μL of PBS buffer, add freshly prepared 0.15 M NaOH for 4 minutes to break the double-stranded bond of the aptamer, and then magnetically separate the supernatant (reverse single-stranded aptamer), then use 100 μL The PBS buffer solution dissolves the binding complex of the magnetic nanoparticles and the forward single-stranded aptamer to complete the preparation of the aptamer purification reagent, which is stored in a refrigerator at 4°C until use.

Purification of unknown biomarkers related to disease in blind samples of positive attributes

In this purification case, the aptamer purification reagent prepared above was used to purify proteins of unknown identity related to diseases with NSCLC EGFR mutations from blind test samples with positive attributes at 22, 31, 177, and 396. In this experiment, the above-mentioned aptamer purification reagents were used to purify unknown biomarkers in serum in a small number of times. Take 100 μL of the serum of the above positive attribute blind test sample (positive attribute sample (1)) and the previously prepared aptamer purification reagent at room temperature and mix thoroughly for one hour. Then perform magnetic separation to remove the supernatant and collect the aptamer. -The magnetic nanoparticles are bound to the complex, washed with PBS buffer several times, and other substances that are not bound but remaining in the wall of the tube are washed away, and then dispersed with 100 μL of PBS buffer, followed by applying a fixed magnetic field strength of 14 gauss (H ₀ ), an initial rotating magnetic field with a magnetic field frequency of 3 KHz (f ₀ ), and disperse the deposited clusters of magnetic particles with a pipette every 2.5 minutes as described above, react for 10 minutes, and collect the magnetic separation The supernatant containing the portion detached from the aptamer-magnetic nanoparticle complex and retaining the aptamer-magnetic nanoparticle binding complex is subjected to a purification procedure by applying a rotating magnetic field. As shown in FIG. 2, the initial rotating magnetic field (f ₀ , H ₀ ) is mainly used to exclude biomolecules that bind to the aptamer with non-specific and low binding affinity. In this embodiment, a part of the supernatant is also used Retained and presented in the subsequent electrophoresis to show that this step can exclude a large number of interfering biomolecules in samples with positive attributes, so as to facilitate subsequent purification and mass spectrometry analysis. Disperse the retained aptamer-magnetic nanoparticle binding complex in 100 μL of PBS buffer, and then apply the next round of rotating magnetic field with a fixed magnetic field strength of 14 gauss (H ₁ ) and a magnetic field frequency of 6 KHz (f ₁ ) , And disperse the deposited magnetic particle clusters with a pipette every 2.5 minutes as described above, react for 10 minutes in total, and magnetically separate and collect the supernatant containing the part detached from the aptamer-magnetic nanoparticle complex ( Separate the component (1)), and retain the aptamer-magnetic nanoparticle binding complex, while dispersing the aptamer-magnetic nanoparticle binding complex in 100 μL of PBS buffer as a sample for the same positive property ( 1) A sample of the purification procedure in which the rotating magnetic field (f ₂ , H ₂ ) is applied in the next round. Repeat the same purification procedure (field conditions are: 6 K, 9 K, 12 K, 15 K, 18 K, 21 K, 24 K, 27 KHz; 14 gauss) and collect each separated component (m, m = 1 to M(= 8)), after the last round of (M) purification of the sample with the same positive property (1), the aptamer-magnetic nanoparticle binding complex retained by magnetic separation is dispersed in 100 μL of PBS buffer It can continue to be used as an aptamer purification reagent to repeat the purification procedure for the next positive attribute sample (2) until all positive attribute samples (n, n = 1 to N) have completed the purification procedure. After completing the purification procedure for all positive attribute samples (n), each set of separated components (m) collected from each positive attribute sample (n) will be collected, and finally Microsep™ Advance Centrifugal Devices (MCP003C41) will be used. 3K, PALL, Taiwan) concentrated the separated components (m) in each group separately for subsequent colloidal electrophoresis of polypropylene amide. The design of the purification procedure is shown in Figure 2.

Polyacrylamide gel electrophoresis of unknown proteins

For this electrophoresis, the casting gel and electrophoresis device of the CAVOY MP-8000 Mini P-4 vertical electrophoresis system (Scientific Biotech Corp., Taiwan) were used. After assembling the device, add a separating gel solution with a specific concentration, and then add an appropriate amount of alcohol to flatten the glue surface. After the separating gel is solidified, pour off the upper layer of alcohol and dry it with filter paper, then add colloid (stacking gel) solution, insert a comb of appropriate size, after the gel is solidified, place it in the electrophoresis tank, and inject the electrophoresis buffer (10x running buffer). The concentrated separated component (m) and the standard molecular weight protein (protein marker) are mixed in a ratio of 3:1 with 5x sample buffer, heated at 98°C for 10 minutes, injected into the tooth groove of the colloid respectively, and the voltage is fixed at Start electrophoresis at 80 volts. When the protein sample moves to the separation colloid, increase the voltage to 100 volts for electrophoresis until the blue dye moves to the bottom of the colloid. Remove the film and place it in deionized water for washing, repeat 3 times, then stain with protein stain (instant blue, GeneMark, Taiwan) for 1 hour, then remove the instant blue solution, and then add deionized water to deionize to deionization After the water does not change color, the distribution of protein bands on the film can be observed, and finally the desired protein size position can be cut out for sequencing. All colloid and buffer components used are shown in Table 5. After collecting the collected supernatant of each frequency interval (containing the protein or biomarker detached from the frequency interval), and then performing electrophoresis on polypropylene amide gel, it can be purified by the virtual filter in each magnetic frequency interval The electrophoresis results of the obtained protein are shown in FIG. 6, and this purification result is the purification result using Aptamer37 for frequency interval scanning. In Figure 6, the first line is protein marker (PM), the second line is to remove a large number of interfering biomolecules (< 3 KHz; 14 gauss), and lines 3 to 9 are separated components (1) (3-6 KHz; 14 gauss) to the separated component (7) (21-24 KHz; 14 gauss), and the last separated component (8) is not juxtaposed because there are no more biomolecules, where the initial magnetic field can be seen in line 2 (Magnetic field conditions: f ₀ = 3 KHz, H ₀ = 14 gauss) A large number of interfering biomolecules can be excluded from the positive attribute samples, while the other lines (lines 3 to 9) appear to use the magnetic field frequency range as a virtual filter It can be used as a biomarker for diseases in which NSCLC has EGFR mutations.

Table 5 Polyacrylamide gel and buffer used in electrophoresis

Sequencing of unknown proteins run by electrophoresis with mass spectrometer

The specific single protein ribbon purified by the aptamer purification reagent was commissioned to AllBio Science Inc. (Taiwan) for mass spectrometry analysis of Bruker autoflex® III MALDI-TOF mass spectrometer (Bruker, Taiwan). In this case, only three protein color bands were selected for mass analysis.

The scanned data of the three purified sample protein color bands indicated by the arrows in FIG. 6 above are shown in FIGS. 7A-7C, respectively. Then compare with the protein database, the result of the first fragment alignment is hypothetical protein I308_05599 (52 KDa), and its alignment score is 82; the result of the second fragment alignment is hypothetical Protein G7K_6558-t1 (58 KDa), its alignment score (score) is 78; the result of the third fragment alignment is ABC transporter (72 KDa), its alignment score (score) is 73.

In general, by using the MARAS platform, aptamers with samples with distinguished NSCLC disease patients with and without EGFR mutations can be selected from the nucleic acid library, and the aptamers obtained by these screenings are further used as capture ligands Purification reagents, using the magnetic field range to provide a virtual filter with a fixed binding affinity range, can be used to purify biomolecules related to the disease from multiple samples with positive attributes, and then confirm the purification by sequencing through a mass spectrometer If the identity of the biomolecule can be further verified with the corresponding monoclonal antibody, it may be found as a biomarker for NSCLC disease with EGFR mutation. The discovery of biomarkers disclosed in the present invention, in addition to the commonly known biochemical related technologies (such as PCR, q-PCR, electrophoresis and mass spectrometry analysis, etc.), there are two main parts: (1) nucleic acid from MARAS platform The library screens out aptamers with specimens that distinguish patients with disease, and (2) uses the scope of the applied oscillating magnetic field as a virtual filter to purify aptamers with captured ligands (screened aptamers) from samples with positive attributes ) Biomolecules with a binding affinity in a certain range. The former considers that the composition of individual patient specimens may be different, so multiple non-disease specimens are used as negative screening samples to remove the nucleic acid library that may be combined with non-disease-related components in non-disease specimens Nucleic acids to reduce the impact of false positives during detection and purification, and multiple positive patient samples are used as samples for positive screening, so that the aptamers obtained by screening can only be shared with all biological samples in positive patient samples Molecular binding to reduce the effect of false negatives in detection and purification, and thus the ability of the obtained aptamers to distinguish samples from patients with and without disease; the virtual filter used in the latter provides capture ligands through the applied oscillating magnetic field (The aptamer obtained by the screening) and the pulling force of the binding pair between the biomolecules. Therefore, when an oscillating magnetic field with an upper and lower cut-off oscillation is applied, the range of the binding affinity of the purified biomolecule and the capture ligand will be from upper to lower. The definition is based on the oscillating magnetic field. If different oscillating magnetic field ranges are used, the range of binding affinity between the purified biomolecules and the capture ligand will be different, that is, different biomolecules will be purified and pass through the samples of patients with multiple positive attributes. The virtual filter is used for purification, and the separated components corresponding to the individual are collected to obtain a sufficient amount of individual biomolecules for sequencing by the mass spectrometer. In addition, the purification technology of the virtual filter regulated by the oscillating magnetic field disclosed in this patent specification can also be used to purify biomolecules (targets) of unknown or known identities from positive attribute samples, and the purified biomolecules The activity will not be reduced by the implementation of purification procedures.

Although exemplary embodiments of the present disclosure have been shown and described above, those of ordinary skill in the art should understand that without departing from the spirit and scope of the present disclosure as defined by the scope of the accompanying patent application, Modifications and changes can be made.

100‧‧‧coil 200‧‧‧Power amplifier 300‧‧‧Signal generator

The drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate embodiments of the present disclosure, and together with the description serve to explain the principles of the present disclosure. FIG. 1 schematically illustrates a screening method for selecting an aptamer having the ability to distinguish a disease patient specimen from a non-patient specimen according to the technique of the present invention using the MARAS method. 2 schematically illustrates the technique according to the present invention, using an aptamer capable of distinguishing between positive and negative specimens as a capture ligand, and using a virtual filter (Virtual Filter) that controls the simulation of the oscillating magnetic field range. Attribute samples are purified from certain disease-related biomolecules. FIG. 3A is a schematic diagram of an experimental setup of a magnetic field-assisted rapid aptamer selection (RO-MARAS) method according to the present invention. 3B is a schematic diagram of an experimental setup of an AC magnetic field magnetic assisted rapid aptamer selection (AC-MARAS) method according to the technology of the present invention. 4A to 4C are three examples of real-time quantitative PCR (q-PCR) for the positive and negative specimens of non-small cell lung cancer patients (NSCLC) with EGFR mutations obtained by MARAS screening according to an embodiment of the present invention. The result of reverse verification by the aptamer. 5A to 5C are three aptamers with the ability to distinguish between positive and negative specimens of EGFR mutant non-small cell lung cancer patients according to an embodiment of the present invention, and blindly analyzing multiple blind test samples via q-PCR the result of. 6 according to an embodiment of the present invention, using Aptamer37 as a capture ligand, from the EGFR mutation of non-small cell lung cancer patients with positive attributes of blindly tested samples of some unknown analytes purified polypropylene amide gel electrophoresis run Glue photo images. FIGS. 7A to 7C illustrate an embodiment of the present invention, depicting a purified sample from a blind test sample of positive attributes of non-small cell lung cancer patients with EGFR mutation using Aptamer37 as a capture ligand through a MALDI-TOF mass spectrometer. Mass spectrogram of mass spectrometry analysis of some unknown analytes (three color bands).

Claims (18)

A method for selecting an aptamer, wherein each of the selected aptamers can distinguish positive samples from negative samples, the method includes: a) providing an oligonucleotide library with random nucleotide sequences as random sequence library, heated and quenched to induce the random sequence library secondary or tertiary structure is formed; b) preparing a plurality of negative samples (bound j) negative samples - magnetic particles _{(NS-MPs (j))} , wherein The magnetic particles (MPs) are nanoparticles (MNPs) or microparticles (MMPs), and where j is a variable integer starting from 1 to J , and each of the J kinds of the negative samples ( j ) is different from other The negative samples are different; c) the random sequence library provided by step a) if j is equal to 1 or the random sequence library provided by step d) of the previous round if j is greater than 1 and one of step b) The negative sample-magnetic particles (NS-MPs _(j) ) are cultured together in the first phosphate buffer (PBS buffer), so that the oligonucleotide and the one negative sample-magnetic particles (NS-MPs _(j)) ) Combination, wherein the random sequence library is cultured with the one negative sample-magnetic particles (NS-MPs _(j) ) one by one or the random sequence library is combined with all different kinds of negative samples-magnetic particles (NS-MPs _{( j)} ) Incubate together in batches; d) Remove the oligonucleotides bound to the negative sample-magnetic particles (NS-MPs _(j) ) by using a magnetic rack for the first magnetic separation and collect the Supernatant of oligonucleotides bound to the negative sample-magnetic particles (NS-MPs _(j) ) to provide step e) when j is equal to J or as a random sequence library when j is less than J Provide the next round of step c) use, where in step c) if the random sequence library is cultured with the one negative sample-magnetic particles (NS-MPs _(j) ) one by one, then the step c) and d) Perform J times, and in the repeating process, each subsequent round of j increases by 1, or if in step c) the random sequence library and all the different kinds of negative samples-magnetic particles (NS-MPs _(j)) with a batch culture, the step c) and d) are performed once; E) preparing a plurality of positive samples in combination with positive samples (i) - the magnetic particles _{(PS-MPs (i))} , and If i equals 1 the supernatant provided by step d) or if i is greater than 1 the supernatant provided by step i) of the previous round and the one positive sample-magnetic particles (PS-MPs _(i) ) Culture together to form a binding mixture containing oligonucleotides bound to the one positive sample-magnetic particle (PS-MPs _(i) ), wherein the magnetic particles (MPs) are nanoparticles (MNPs) Or microparticles (MMPs), and each of the I positive samples ( i ) is different from the other positive samples The same, and i is a variable integer starting from 1 to I ; f) by using the magnetic rack for a second magnetic separation to collect the positive sample containing the same type of magnetic particles (PS-MPs _(i) ) The binding mixture of bound oligonucleotides removes the supernatant containing oligonucleotides that are not bound to the one positive sample-magnetic particle (PS-MPs _(i) ), and makes the collected The positive sample-magnetic particles (PS-MPs _(i) ) bound oligonucleotide binding mixture is re-dispersed in the second phosphate buffer; g) the redispersed obtained from step f) The binding mixture is subjected to a magnetically assisted rapid aptamer selection under a first oscillating magnetic field with a lower frequency f _L and/or a lower intensity H _L , so that the positive sample-magnetic particles (PS-MPs _(i) ) have Oligonucleotides with a binding affinity lower than the first binding affinity are separated from the one positive sample-magnetic particles (PS-MPs _(i) ), and a third magnetic separation is performed by using the magnetic rack to remove The one kind of positive sample-magnetic particles (PS-MPs _(i) ) has a supernatant of the separated oligonucleotide having a binding affinity lower than the first binding affinity to collect the sample containing the one kind of positive sample -The binding mixture of the oligonucleotides to which the magnetic particles (PS-MPs _(i) ) are bound; h) the sample collected from step g) containing the positive sample with the one-magnetic particles (PS-MPs _(i)) ) The binding mixture of the bound oligonucleotide is re-dispersed in the third phosphate buffer; i) subjecting the re-dispersed binding mixture obtained from step h) to an upper frequency f _U and/or an upper intensity H _U The magnetic-assisted rapid aptamer selection under the second oscillating magnetic field is to combine the oligonucleotide with the binding affinity to the positive sample-magnetic particle (PS-MPs _(i) ) lower than the second binding affinity with the The positive sample-magnetic particles (PS-MPs _(i) ) are separated, and a fourth magnetic separation is performed using the magnetic rack to collect the positive sample-magnetic particles (PS-MPs _(i)). ) The supernatant of the separated oligonucleotide having a binding affinity lower than the second binding affinity for step j) if i is equal to I or for the next round of step e) when i is less than I , and Remove the remaining binding mixture containing oligonucleotides with binding affinity higher than the second binding affinity for the one positive sample-magnetic particle (PS-MPs _(i) ), where f _L ≤ f _U and/or H _L ≤ H _U and excluding the simultaneous use of f _U = f _L and H _U = H _L , or elution of the positive sample-magnetic particles (PS -MPs _(i) ) whose binding affinity is higher than the first binding affinity, through the use of The magnetic rack performs a fifth magnetic separation to collect the supernatant containing oligonucleotides having a binding affinity higher than the first binding affinity for the one kind of positive sample-magnetic particles (PS-MPs _(i) ) Liquid to be used for step j) if i is equal to I or for the next round of step e) when i is less than I , wherein the steps e) to i) are repeated I times, and in the repeating process, each round of i is increased by 1, Or if the components of the positive sample are fixed and known, then the steps e) to i) are performed once, and wherein the second binding affinity is higher than the first binding affinity; and j) obtained with the ability to distinguish The oligonucleotides of the positive sample and the negative sample are used as aptamers. The method according to item 1 of the patent application scope, wherein step b) includes: providing J negative samples ( j ), and combining the J negative samples ( j ) with a plurality of the magnetic particles to form J Negative sample-magnetic particles (NS-MPs _(j) ); and step e) includes: providing 1 positive sample ( i ), and combining the 1 positive sample ( i ) with a plurality of the magnetic particles positive sample to form Type I - magnetic particles _{(PS-MPs (i))} . The method according to item 2 of the patent application scope, wherein the method of combining the J- negative samples ( j ) or the I- positive samples ( i ) with the magnetic particles, respectively, includes: The pair of the magnetic particles and the J negative samples ( j ) or the I positive samples ( i ) joins the J negative samples ( j ) or the I positive samples ( i ) to the Magnetic particles. The method according to item 3 of the patent application scope, wherein the conjugated pair is composed of streptavidin and biotin, the streptavidin is bound to the magnetic particle, and the biotin is The J negative samples ( j ) or the I positive samples ( i ) are combined. The method according to item 1 of the patent application scope, wherein step g) subjects the redispersed binding mixture to a magnetically assisted rapid aptamer under a first oscillating magnetic field having a lower frequency f _L and/or a lower intensity H _L The method of selection includes: performing the selection of the magnetic-assisted rapid adaptor by applying a first rotating magnetic field or a first alternating magnetic field having the lower frequency f _L and/or the lower intensity H _L. The method according to item 1 of the patent application scope, wherein step i) subjecting the redispersed binding mixture to a magnetically assisted rapid aptamer under a second oscillating magnetic field having an upper frequency f _U and/or an upper intensity H _U The method of selection includes: performing the magnetic-assisted rapid aptamer selection by applying a second rotating magnetic field or a second alternating magnetic field having the upper-limit frequency f _U and/or the upper-limit intensity H _U. A method for purifying at least one type of target biomolecule from a positive sample using an aptamer, the aptamer can distinguish the positive sample from a negative sample and can be the same as the at least one type of target biomolecule in the positive sample In combination, the method includes: a) providing the aptamer, wherein the aptamer is dispersed in a first phosphate buffer (PBS buffer), and is heated and annealed to induce secondary or tertiary structure Forming; b) providing N positive samples ( n ), wherein each of the positive samples contains the at least one type of target biomolecule, and n is a variable integer starting from 1 to N ; c) preparing multiple magnetic particles and The aptamer in step a) is combined to form an aptamer-magnetic particle binding complex and used as a purification reagent to purify the at least one type of target biomolecule from the positive sample ( n ), wherein The magnetic particles (MPs) are nanoparticles (MNPs) or microparticles (MMPs); d) the purification reagent provided by step c) if n equals 1 or step i of the previous round if n is greater than 1 ) The provided purification reagent is incubated with a positive sample ( n ) of step b) to form a magnetic particle-aptamer-biomolecule binding mixture during the purification process of the at least one type of target biomolecule; e ) Subjecting the magnetic particle-aptamer-biomolecule binding mixture in step d) to an initial (0th) oscillating magnetic field with an initial frequency f ₀ and/or an initial intensity H ₀ , so that the binding affinity is lower than Biomolecules initially (0th) binding affinity bound to the aptamer are detached from the one kind of magnetic particle-aptamer-biomolecule binding mixture, including binding to the aptamer with non-specific and low binding affinity The biomolecules; f) collect the remaining one kind of magnetic particles-aptamer-biomolecule binding mixture in step e) by using a magnetic rack for the first magnetic separation to remove the one containing the one kind of magnetic particles- The supernatant of the biomolecule or unbound biomolecule detached from the aptamer-biomolecule binding mixture, and redispersing the collected one kind of magnetic particle-aptamer-biomolecule binding mixture in the second In phosphate buffer; g) a redispersed magnetic particle-aptamer-biomolecule binding mixture provided by step f) if m is equal to 1 or provided by step h) of the previous round if m is greater than 1 The redispersed magnetic particle-aptamer-biomolecule binding mixture is subjected to the (m)th oscillating magnetic field having the (m)th frequency f _m and/or the (m)th intensity H _m so that the binding affinity is high The biomolecule bound to the aptamer at the (m-1)th binding affinity and lower than the (m)th binding affinity is detached from the one kind of magnetic particle-aptamer-biomolecule binding mixture, wherein the ( m-1) The binding affinity is lower than the (m)th binding affinity, and m is a variable integer starting from 1 to M ; h) by using the The magnetic rack performs the second magnetic separation to collect the remaining one kind of magnetic particle-aptamer-biomolecule binding mixture, and retains the one containing one kind of magnetic particle-aptamer-biomolecule binding mixture from step g). The supernatant of the detached biomolecules is used as the separation component ( m ) to provide step j), and the collected magnetic particle-aptamer-biomolecule binding mixture is re-dispersed in the third phosphate In the buffer for step i) if m is equal to M or for the next round of step g) when m is less than M , where steps g) to h) are repeated M times, and in the repeat process, m increases for each round 1, or if the oscillating magnetic field range (f _(m-1) , H _(m-1) , f _(m) , and H _(m) ) for a particular type of target biomolecule is known, then the Steps g) to h) are performed once until all biomolecules in the one magnetic particle-aptamer-biomolecule binding mixture are completely separated from the aptamer, and the one magnetic particle-aptamer-biological The molecular binding mixture is restored to an aptamer-magnetic particle binding complex, and where f _(m-1) ≤ f _(m) and/or H _(m-1) ≤ H _(m) and the simultaneous use of f _{(m )} = f _(m-1) and H _(m) = H _{(m -1)} ; i) A aptamer-magnetic particle binding complex collected and re-dispersed as described in step h) is used as a purification reagent or Purify the purification reagent according to the method of step c) to purify the at least one type of target biomolecule in the next positive sample ( n ), and repeat the steps d) to i) N rounds, and During the repetition process, n increases by 1 each round, and the conditions of the oscillating magnetic field applied in each round remain unchanged; and j) collect the steps h) obtained by sequentially performing the steps d) to i) of N rounds separation of corresponding types of components m (m), to obtain a purified m types of target biological molecule. The method according to item 7 of the patent application scope, wherein step c) preparing a plurality of the magnetic particles and the aptamer to form the aptamer-magnetic particle binding complex includes: The aptamer is provided and the aptamer is combined with the magnetic particles via a bonding pair to form the one kind of aptamer-magnetic particle binding complex, wherein the bonding pair is composed of streptavidin and It is composed of biotin, and the streptavidin binds to the magnetic particles, and the biotin binds to the aptamer. The method according to item 7 of the patent application scope, wherein step e) a method of subjecting the magnetic particle-aptamer-biomolecule binding mixture to an initial oscillating magnetic field having an initial frequency f ₀ and/or an initial intensity H ₀ The method includes: performing magnetic assisted removal by applying an initial rotating magnetic field or an initial alternating magnetic field having the initial frequency f ₀ and/or the initial intensity H ₀ , the magnetic force assisted removal is from the one type of magnetic particles -The aptamer-biomolecule binding mixture removes biomolecules that bind to the aptamer with a binding affinity lower than the initial binding affinity, including those that bind to the aptamer with non-specific or low binding affinity The biomolecule. The method according to item 7 of the patent application scope, wherein step g) subjecting the re-dispersed magnetic particle-aptamer-biomolecule binding mixture to a frequency (m) f _m and/or a (m) The method of the (m)th oscillating magnetic field with the intensity H _m includes: by applying the (m)th rotating magnetic field or the (m)th with the (m)th frequency f _m and/or the (m)th intensity H _m An alternating magnetic field is used to perform magnetic-assisted removal such that the binding affinity is higher than the (m-1)th binding affinity and lower than the (m)th binding affinity. The biomolecule of the aptamer is detached from the magnetic particle-aptamer-biomolecule binding mixture. A method for purifying at least one type of target biomolecule from a positive sample with a ligand, and the ligand can distinguish the positive sample from a negative sample and can bind to the at least one type of target organism in the positive sample Molecule, the method includes: a) providing the ligand, and dispersing the ligand in a first phosphate buffer (PBS buffer) as a purification reagent to provide for purification from the positive sample Out the at least one type of target biomolecule; b) provide N positive samples ( n ), wherein each of the positive samples ( n ) contains the at least one type of target biomolecule, and n is from 1 to N Variable integer; c) one of the positive samples ( n ) of step b) and the purification reagent provided by step a) if n is equal to 1 or the purification provided by step g) of the previous round if n is greater than 1 The reagents are cultured together to form a ligand-biology through the action of at least one type of conjugation pair between the ligand and the at least one type of target biomolecule in the one positive sample ( n ) A molecular binding mixture; d) subjecting the one ligand-biomolecule binding mixture of step c) to a virtual filter having a binding affinity capable of selecting the at least one type of binding pair to remove from the one ligand-biological In the molecular binding mixture, remove unbound and non-specifically bound biomolecules with a binding affinity of the binding pair formed with the ligand that is lower than the initial (0th) binding affinity, and collect the remaining one ligand-biomolecule A binding mixture, wherein the virtual filter has the ability to change the binding affinity range selection for the at least one type of binding pair; e) the one ligand-biomolecule provided by step d) if m is equal to 1 The binding mixture or the ligand-biomolecule binding mixture provided by step f) of the previous round if m is greater than 1 is subjected to the virtual filtering with the mth binding affinity selected above the (m-1)th binding affinity To separate and collect the m- th type target biomolecule from the one ligand-biomolecule binding mixture to provide step h), and retain the remaining one ligand-biomolecule binding mixture to provide step f) Using, wherein the conjugated pair formed by the m- th type target biomolecule and the ligand has a binding affinity higher than the (m-1)th binding affinity and lower than the (m)th binding affinity, And the (m-1)-th binding affinity is lower than the m-th binding affinity; f) gradually increasing the ratio of the ligand-biomolecule binding mixture selected by the virtual filter to step e) The binding affinity of the at least one type of conjugated pair formed between the body and the at least one type of target biomolecule, and repeating steps e) to f) M rounds, and in the repeating process, m increases by 1 for each round, or If the ligand in the ligand-biomolecule binding mixture is combined with a single specific type of target biomolecule If the applicable range of the binding affinity of the inter-zygosity pair is known, the virtual filter with the applicable range of the binding affinity of the conjugation pair is subjected to the rounds of steps e) to f) until all All the biomolecules in the ligand-biomolecule binding mixture are completely separated, and the ligand-biomolecule binding mixture is restored to the ligand; g) the ligand obtained in step f) is re-dispersed Use the second phosphate buffer as the purification reagent or prepare the purification reagent according to the method of step a) to purify at least one type of target biomolecule in the next positive sample ( n ), and repeat the steps c) to g) N rounds, and in the repeating process, n is increased by 1 in each round, and the conditions of the virtual filter used for the purification of each positive sample ( n ) in each round remain unchanged; and h ) Collect the M types of corresponding target biomolecules obtained from the step c) to step e) of N rounds in sequence to obtain M types of purified target biomolecules. The method according to item 11 of the patent application scope, wherein the ligand-biomolecule binding mixture in step c) comprises at least one type of junction formed by the ligand and the at least one type of target biomolecule Yes, and wherein the binding affinity formed by the ligand and the at least one type of each type of target biomolecule is different from other types of binding affinity. The method according to item 12 of the patent application scope, wherein the at least one type of conjugated pair includes the following pairs: antibody-antigen, DNA/RNA aptamer-captured biomolecule, and single-stranded DNA and its complementary strands. The method according to item 11 of the patent application scope, wherein in step d), the one ligand-biomolecule binding mixture is subjected to the ability to select the binding affinity range of the at least one type of binding pair The virtual filter, and wherein the selection mechanism of the virtual filter includes applying mechanical force, hydrodynamic force, centrifugal force, electromagnetic force, or any combination thereof to the one ligand-biomolecule binding mixture. The method according to item 11 of the patent application scope, wherein the purification reagent using the ligand as the purification method for purifying at least one type of the target biomolecule from the positive sample includes: The ligand is bound to the immobilization surface as a solid support of the purification method for purifying the at least one type of the target biomolecule from the positive sample. The method according to item 11 of the patent application scope, wherein the purification reagent using the ligand as the purification method for purifying the at least one type of the target biomolecule from the positive sample includes: Providing a plurality of magnetic particles or dielectric particles, and causing the ligand and the magnetic substance suspended in the solution in the purification method of purifying the at least one type of the target biomolecule from the positive sample The particles or the dielectric particles are combined, and wherein the magnetic particles or the dielectric particles are nano particles or micro particles. The method according to item 11 of the patent application scope, wherein the solution and the solution in the purification method of suspending the ligand and suspending the at least one type of the target biomolecule from the positive sample The combination of the magnetic particles or the dielectric particles in includes: The magnetic particles or the dielectric particles bound to the ligand are collected through a collection method. The method as described in Item 17 of the patent application range, wherein the one collection method includes a geometric capture method or a capture method through a gradient magnetic field or a gradient electric field.

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